PROJECT SUMMARY A synthetic lethality screen was performed to identify hyper-dependencies in melanomas harboring deficiencies in ARID genes?components of the SWI/SNF chromatin remodelers?which occur in 20-30% of human cancers. ARID mutant melanomas exhibited extreme dependency on the H3K9-dimethylase G9a, a druggable enzyme. This led to discovery of 6 melanoma cases harboring recurrent point mutation at an identical residue in G9a?s catalytic domain. The mutation hyper-activates G9a and aligns to oncogenic mutations in EZH2, a previously known oncogenic methyltransferase that targets a different histone-3 lysine (K27). Beyond these rare activating G9a mutations we identified more common genomic G9a copy gains in 27.8% of melanomas in TCGA and found G9a dependency (via genetic or pharmacologic means) in human melanoma lines with as little as one extra G9a copy. Corroborating our results, the Achilles Project (genome- scale loss-of-function viability screening in many deeply annotated cancer cell lines) confirmed G9a dependency among melanoma lines harboring either G9a amplification or ARID deficiency. The same dependencies were also seen in multiple cancers beyond melanoma in Achilles. Furthermore, G9a copy gain was accompanied by global elevations in H3K9 dimethylation, which correlated as a biomarker of G9a dependency in G9a gained/amplified melanoma cells. Mechanistic studies revealed activation of the canonical Wnt pathway as a vital dependency target, which is induced by G9a?s suppression of Wnt antagonist DKK1. Tumor intrinsic Wnt activation is common in melanomas, though usually without pathway-intrinsic mutations?a phenomenon now largely explained by G9a activation. Importantly, Wnt activity has been strongly implicated by others as a trigger of tumor-induced immune evasion?of keen interest for efficacy of immunotherapy. Here, Aim 1 will validate dependencies of G9a copy-gained and ARID loss-of-function human melanoma lines on G9a, GLP, MITF, DKK1, and Wnt signaling. Global ATAC-seq, RNA-seq, and H3K9me2-ChIP-seq will be used to identify additional G9a targets and mediators of viability in these melanomas. Aim 2 will test pharmacologic G9a inhibition in multiple mouse and zebrafish models containing copy gains of G9a or GLP, or ARID2 deficiency, combined with either BRAF-targeted therapy or anti-PD1 immunotherapy. The latter study will utilize novel isogenic engineered syngeneic murine melanomas containing or lacking UV-neoantigens, permitting measurements of G9a targeting as a strategy to blunt the Wnt pathway?s immunosuppressive activity, together with direct targeting of G9a?s cell-intrinsic oncogenic function. Human melanoma biopsies will also be interrogated to corroborate preclinical models. These studies identify a novel oncogene (G9a) and, through vital collaborations with Dr. Zon (zebrafish melanoma model), Drs. Liu and Zon (epigenetic mechanisms), and Drs. Wucherpfennig and Rodig (immunological profiling), will reveal mechanisms, novel biomarkers, and therapeutic opportunities in melanoma, with relevance to other treatment-resistant cancers.